KR101032956B1 - Rapid diagnostic kit of hemorrhagic fever with renal syndrome detecting specific IgM and IgG using nucleocapsid protein derived from Soochong virus - Google Patents

Abstract

The present invention provides a diagnostic composition for Hemorrhagic fever with renal syndrome (HFRS) comprising a nucleocapsid protein (NP) derived from Soochong virus and antigen-specific IgM and IgG using the same. A rapid diagnostic kit for detecting.

Hearing virus, nephrotic bleeding, immunochromatography

Description

Rapid diagnostic kit of hemorrhagic fever with renal syndrome detecting specific IgM and IgG using nucleocapsid protein derived from Soochong virus}

The present invention provides a composition for diagnosing hemorrhagic fever with renal syndrome (HFRS) using a nucleocapsid protein (NP) derived from a Soochong virus, and rapidly using antigen-specific IgM and IgG. It relates to a quick diagnostic kit for detecting.

Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantavirus (Hantavirus), which causes 20-300,000 patients worldwide every year, and 3-7% of them die of acute febrile disease in Korea. Thousands of patients per year are classified as Class 2 legal epidemics that occur in civilians and soldiers.

The genus Hantavirus virus is a species of Hantan virus, Seoul virus, Puumala virus and Prospect Hill virus, and recently, Soochong. virus) has been isolated.

Hantan virus is a prototype virus of HFRS. It is a virus adapted to rats by inoculating peripheral blood from a Korean Hemorrhagic Fever (KHF) patient inoculated with Apodemus agrarius . Hantan virus is distributed throughout the world, including Korea, the Soviet Union, Manchuria, and Europe, and is known to be the most pathogenic in humans. The clinical course takes about two to four months after infection through five stages of fever, hypotension, urination, diuresis and recovery.

Seoul virus rats (Rattus norvegious ), isolated from the lung tissue, neutralizes and differs from the Hantan virus, and is pathogenic to humans, weaker than the Hantan virus, causing moderate renal hemorrhagic fever. Many patients with nephrotic bleeding hemorrhagic fever in the city are infected with Seoul virus, and many cases have been reported in Korea, Japan and Southeast Asia.

Fumala virus is mainly distributed in Europe, mainly in Sweden, with mild clinical symptoms, little bleeding, and renal symptoms. In addition, it is difficult to distinguish the five stages that can be observed in patients with typical hemorrhagic fever.

Prospect Hill virus is known to be nonpathogenic in humans. Hepatitis virus is currently being studied to determine its etiology.

Nephrotic bleeding hemorrhagic fever is similar to the symptoms of the flu or in the case of intractable infections in addition to the typical clinical characteristics, and due to the problem of differentiating between febrile infectious diseases such as Tsutsugamus disease and leptospirosis in Korea Substantial diagnosis is dependent on the results of serological tests in addition to clinical findings. The serological diagnostic method for nephrotic bleeding hemorrhagic fever is the most commonly used indirect immunofluorescence assay (IFA), but there may be false judgments made by subjective readings. The process is very cumbersome and takes more than four hours. In addition, enzyme linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), plaque reduction neutralization test (PRNT), western blot analysis (WB), reverse transcription Although various assays, such as polymerase chain reaction (RT-PCR), are used, antigen test methods of high reliability, which are important for the determination of disease progression, the degree of treatment, and the prognosis of patients, still have problems to be improved.

Recently, there are four methods for producing a diagnostic kit that are widely used, such as Enzyme Immunoassay (EIA), Aggregation Assay, Dip-stick, and Lateral Flow Assay. EIA has the advantage of having high sensitivity, but it has a disadvantage in that it takes a lot of time and requires special reading equipment because the specificity is low and many pretreatment steps are required before diagnosis. Plate agglomeration also requires a pretreatment process, but the short time required for reading is low, while the sensitivity is low and the results are not easy to read. Tube agglomeration is often cumbersome and ambiguous in reading and time consuming. It has the disadvantage of taking more time. Dip-stick (Dip-stick) has the advantage of being applicable to a variety of pathogens and low cost, but also has the disadvantage of requiring a pretreatment process and takes about 3 hours to read. Lateral flow assay method, also called rapid diagnostic kit, is the most recently developed method because it can be diagnosed in a short time. It does not require pretreatment and can be read visually within 15 minutes. The application has the disadvantage of securing a specific antigen for a specific pathogen.

Thus, the present inventors have found that the sensitivity and specificity of nephrotic bleeding hemorrhagic fever can be improved by detecting antigen-specific IgM and IgG using nucleocapsid protein of the anti-virus of Hantavirus. It was.

One object of the present invention is to provide a composition for diagnosing hemorrhagic fever with nephrotic bleeding, comprising a nucleocapsid protein derived from a bovine virus having an amino acid sequence of SEQ ID NO: 2 or 3.

Another object of the present invention is to provide a kit for diagnosing nephrotic bleeding fever using the nucleocapsid protein and a method for detecting an antibody against nephrotic bleeding fever using the same.

In one embodiment, the present invention relates to a composition for diagnosing hemorrhagic fever with nephrotic bleeding, which comprises a viral-derived nucleocapsid protein having an amino acid sequence of SEQ ID NO: 2 or 3.

As used herein, the term “diagnosis” refers to identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to identify nephrotic bleeding fever.

In the present invention, the term "nephrotic bleeding hemorrhagic fever" refers to a disease caused by hantan virus and Seoul virus. In the present invention, it is determined whether hepatitis hemorrhagic fever is infected with a hepatitis virus-derived nucleocapsid protein (CNP) having an amino acid sequence of SEQ ID NO: 2 or a hepatitis virus-derived nucleocapsid protein fragment (TNP) having an amino acid sequence of SEQ ID NO: 3; Diagnosis can be made.

Hepatitis virus is a virus belonging to Hantavirus and has only recently been discovered. Hantaviruses have three negative-stranded RNAs as genomes and can be classified into L (large), M (middle), and S (small) according to their size. The L segment contains genes for the virus-specific RNA dependent RNA polymerase, and the M segment contains glycoprotein 1 (G1), a membrane protein of 65 to 74 and 55 to 60 kDa in size. It has a gene for glycoprotein 2 (G2), and the S segment has a gene for a nucleocapsid protein (NP) of 50 to 53 kDa size that surrounds each segment.

The inventors have found that the nucleocapsid protein (CNP) having the amino acid sequence of SEQ ID NO: 2 from the S segment (SEQ ID NO: 1) and the fragment thereof, the protein fragment of the nucleocapsid having the amino acid sequence of SEQ ID NO: 3 (TNP) 1) and confirmed that they react with the sera of patients with nephrotic bleeding fever but do not react with normal serum, and thus can be usefully used as antigens for diagnosing nephrotic bleeding.

The protein having the amino acid sequence of SEQ ID NO: 2 or 3 may be chemically synthesized or may be prepared in microorganisms or eukaryotic cells using a genetic recombination method, but is not necessarily limited thereto. As a vector for using the genetic recombination method, it is preferable to use a transfer vector capable of expressing a recombinant protein without infecting humans and animals. In a specific implementation of the invention, baculovirus transfer vectors were used.

As another aspect, the present invention relates to a nephrotic hemorrhagic fever diagnostic kit comprising a test strip comprising the antigen as a antigen of a nucleocapsid protein derived from the amino acid sequence of SEQ ID NO: 2 or 3.

Specifically, the present invention provides

(a) a sample pad on which a sample is absorbed;

(b) gold conjugation pads that bind to human antibodies in the sample;

(c) A test line in which a test line comprising a receptor-derived nucleocapsid protein having an amino acid sequence of SEQ ID NO: 2 or 3 as an antigen and a control line comprising a control protein are treated. membrane); And

(d) Absorption pads in which the remaining sample is absorbed

It relates to a nephrotic bleeding fever diagnostic kit for detecting nephrotic bleeding fever antibodies (IgM and IgG) in a biological sample consisting of a test strip comprising a.

As shown in FIG. 8, the preferred nephrotic bleeding fever diagnostic kit of the present invention includes a test strip including a sample pad, a gold binding pad, a reaction membrane, and an absorption pad. The sample pads are superimposed on the gold bond pads to form a first overlapping portion, and the gold bond pads are superimposed on the reaction film to form a second overlapping portion. In addition, the reaction film and the absorption pad form a third overlapping portion. When the biological sample is deposited on the sample pad, the capillary phenomenon causes the biological sample to be added to a gold binding pad, eg, gold-labeled protein A or gold-labeled goat anti human IgM antibody. They pass through a gold binding pad containing goat anti-human IgM antibody. The gold particles forming the gold-label preferably have a diameter size of 20 to 55 nm, more preferably a diameter size of 20 to 40 nm, and serve as an indicator dye. As it passes through the gold binding pad, the antibody in the biological sample binds to the gold-labeled protein A or the like to form a complex, and the complex moves along the reaction membrane.

As used herein, the term "biological sample" or "sample" refers to serum, plasma, blood, and the like, of mammals including humans suspected of having nephrotic bleeding. Biological samples can be used with or without dilution prior to dropping onto the sample pad of the diagnostic kit of the present invention. If the biological sample contains antibodies against hantan virus or Seoul virus, the color change in the reaction line on the reaction membrane can be visually combined with nucleocapsid protein antigens derived from the reception virus in the diagnostic kit of the present invention. Happens. However, if the biological sample does not have an antibody against Hantan virus or Seoul virus, it does not bind with the antigen of the present invention, and thus no change occurs in the reaction line on the reaction membrane. In other words, in a preferred embodiment of the present invention, the hantanvirus antibody binds to a nucleocapsid protein antigen derived from a hearing virus immobilized on a reaction line on the reaction membrane. Formation of this antigen-antibody complex results in a reddish purple band that can be detected as the indicator dye by the gold complex precipitates, thus a positive reaction of the presence of an antibody against Hantan virus or Seoul virus in a biological sample. Will be displayed.

The reaction membrane may comprise a suitable material such as a nitrocellulose membrane (Milipore ^™ XA3J072100) of standardized size (10 × 500 nm). The reaction membrane is preferably arranged in the order of the reaction line and the control line, these reaction line and the control line may be arranged at intervals of 2.0 to 4.5mm, preferably 2.5 to 3.0mm.

The result of a positive response to nephrotic bleeding hemorrhagic fever is a case where a reaction line containing an antigen protein having an amino acid sequence of SEQ ID NO: 2 or 3 in addition to the control line develops. The control line is for confirming the abnormality of the diagnostic kit. The negative response to nephrotic bleeding hemorrhagic fever was in the control group. The control line contains a control protein that binds to the gold-label, for example rabbit anti-goat IgG polyclonal antibody, goat anti-human IgG polyclonal antibody, rabbit anti-human IgM polyclonal antibody or goat anti- Human IgM polyclonal antibodies, and the like, and these control proteins may be included at a concentration of 0.1 mg / ml to 1.0 mg / ml. The reaction line comprises a recipient virus-derived nucleocapsid antigen protein having an amino acid sequence of SEQ ID NO: 2 or 3, and these antigen proteins are included in the reaction line at a concentration of 0.5 to 1.0 mg / ml.

Gold binding pads are suitable markers such as dried colloidal gold-labeled protein A, gold-labeled anti-human IgG antibody or gold as a marker. -Labeled anti-human IgM antibodies and the like, and may preferably be located adjacent to the sample pad. When the gold-labeled marker is included in the gold binding pad in an excessive amount, the antibody against the anti-tantan virus to be analyzed may be increased to a non-specific signal even in a relatively low region, resulting in a false positive result. The optical density (OD) may be included in a concentration of 1 to 6, preferably 2 to 4 absorbance. Absorption pads are located at opposite ends of the membrane and absorb biological samples such as serum, plasma, etc. that have migrated along the membrane by capillary action.

In one specific implementation, the diagnostic time is about 5-10 minutes, which is within 15 minutes. Diagnostic results can be used to diagnose Hantavirus infection status in patients. For example, if the biological sample collected from a patient with nephrotic bleeding fever, the diagnostic kit of the present invention may be positive for diagnosing nephrotic bleeding fever, and if the biological sample collected from a patient without nephrotic bleeding fever. The diagnostic kit of the invention can be diagnosed as not having a nephrotic bleeding fever with a negative response.

The sensitivity and specificity of the hemorrhagic fever of nephrotic syndrome in a diagnostic kit containing the above-mentioned antivirus-derived nucleocapsid antigen protein having the amino acid sequence of SEQ ID NO: 2 or 3 were compared with the indirect immunofluorescence antibody (IFA). , The diagnostic kit of the present invention showed a sensitivity and specificity of more than 96%, even in the initial diagnosis of detecting IgM showed more than two times higher sensitivity than the indirect immunofluorescent antibody method. Therefore, the diagnostic kit of the present invention can effectively diagnose nephrotic bleeding hemorrhagic fever, and in particular, the initial infection can be diagnosed more effectively than the conventional method.

In another embodiment, the present invention relates to nephrotic hemorrhagic fever consisting of obtaining a biological sample from a patient suspected of nephrotic bleeding fever and detecting an antibody from the biological sample using the nephrotic hemorrhagic fever diagnostic kit. It relates to an antibody detection method.

Specifically, if a biological sample, for example, serum is collected from a patient suspected of nephrotic bleeding fever, and the collected biological sample is dropped onto a sample pad which is a site where a sample of the diagnostic kit is absorbed, The capillarity causes the gold conjugation pad to reach the gold conjugation pad so that the antibody in the sample is labeled with a marker in the gold binding pad, e.g., gold-labeled protein A, gold-labeled anti-human IgG. It forms a colloid by binding to an antibody (gold-labeled anti-human IgG antibody) and a gold-labeled anti-human IgM antibody. In this case, the biological sample to be sampled continues to move without being immobilized on the gold binding pad to reach a test line in which the anti-virus-derived nucleocapsid antigen protein is immobilized. In biological samples of patients with nephrotic hemorrhagic fever, an antigen-antibody reaction with the antigen occurs, i.e., an antibody bound to a specific marker in the gold binding pad of the patient is directed to a nucleocapsid antigen protein derived from an antiviral strain immobilized on the response line. By combining, they form a reddish purple band that can be visually observed. In addition, the markers in the remaining gold binding pads that did not react with the antibodies in the biological sample reached the control line to react with the control protein to form a red purple band, indicating the suitability of the test. By such a method, antibodies to nephrotic bleeding hemorrhagic fever can be detected.

As described above, the composition for diagnosing nephrotic bleeding fever of the present invention and the diagnostic kit using the same provide improved sensitivity and specificity compared to the conventional diagnostic method, and can easily and quickly diagnose nephrotic bleeding fever.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, this is only added to help the understanding of the present invention, the scope of the present invention is not limited by these examples.

Example  One: Hearing virus  And cultured cells

Soochong virus strain SooV-2 isolated from domestic Apodemus peninsulae was passaged in Vero cells. Proliferation and maintenance of Vero cells were incubated in a 37 ° C incubator with 5% CO ₂ in EMEM (Eagel's minimal essential medium) containing 10% fetal bovine serum (FBS). It was used when the degree of monolayer culture.

Example 2: Isolation of Viral RNA

After centrifuging the cells in which the virus was propagated at 1,075g (3000rpm) for 3 minutes, 750ul of Trizol LS (Gibco) solution was added and allowed to stand at room temperature for 5 minutes, followed by adding 200ul of chloroform and mixing well. It was left for 10 minutes. The precipitate was removed by centrifugation at 17,000 g (12,000 rpm) at 4 ° C. for 15 minutes, and the same volume of isopropyl alcohol was added to the supernatant and allowed to stand at room temperature for 10 minutes, then again at 17,000 g (12,000 rpm) at 4 ° C. RNA was precipitated by centrifugation for 15 minutes and washed once with 75% ethanol. The RNA precipitate was dried in air for 10 minutes and then dissolved in DEPC treated water.

Example 3: cDNA Synthesis and Chain Enzyme Polymerization

1 ul of RNA solution isolated in Example 2 to synthesize cDNA from the nucleic acid sequence of SEQ ID NO: 1 (GenBank accession No. AY675350) encoding the nucleocapsid protein (NP) of FIG. Each primer (10 pmole) 1 ul, 5X reverse transcriptase buffer), 10mM dNTPs, 100pmole / ul Oligo (dT), 40 units / ul RNasin, 200 units / ul MMLV reverse transcriptase mixture was added. After reacting at 30 ° C. for 10 minutes, cDNA was synthesized by reacting at 45 ° C. for 30 minutes, 99 ° C. for 5 minutes, and 5 ° C. for 5 minutes.

PCR was performed by mixing 5 cul of cDNA product, a pair of 10 pmole primer, 10 mM dNTPs, 10X taq buffer, 5 units / ul Taq polymerase, and then reaction once every 95 ° C for 5 minutes, 52 ° C for 2 minutes, 72 ° C for 2 minutes, and 95 ° C for 1 minute. The reaction was carried out five times at a cycle of 52 ° C. for 1 minute and 72 ° C. for 1 minute. 95 ° C 1 minute, 60 ° C 1 minute. The reaction was carried out 20 times at 72 ° C. for 1 minute, and DNA amplification was performed once at 95 ° C. for 1 minute, 60 ° C. for 1 minute, and 72 ° C. for 7 minutes. Amplified DNA was stored at -20 ° C.

As shown in FIG. 2, PCR was performed with each primer to confirm DNA of fragment protein (Truncated NP: TNP) at about 370 bp, and DNA of whole protein (Complete NP: CNP) was also identified as a band at about 1,300 bp.

PCR primers for the production of Soochong Virus recombinant antigens Primer Whole protein (CNP) Fragment Protein (TNP) Forward
primer 5 ' AGA TCT A ATG GCA ACT ATG GAG GAA TTG 3 '(SEQ ID NO: 4)
Bgl Π (AGATCT); AGATCT Added> 5 ' AGA TCT A ATG GCA ACT ATG GAG GAA TTG 3 '(SEQ ID NO: 6)
<Bgl Π; AGATCT Added> Reverse
primer 5 ' AAGC TTA TAG TTT TAA AGG TTC TTG GTT TG 3' (SEQ ID NO: 5)
Hind III (AAGCTT); AAGC addition> 5 ' A AGC TTA CCA GTC TGC AGT CTG ACC TGT 3 '(SEQ ID NO: 7)
<Hind III; Add AAGCTTA>

Example 4: Cloning of Amplification Products

About 370 bp and about 1,300 bp of DNA amplified by the chain enzyme polymerization reaction were electroeluted in agarose gel and cloned into the cloning vector pGem-T-Easy PCR cloning vector (Invitrogen) (FIG. 3). DNAs of TNP and CNP were identified by digestion with restriction enzymes Bgl Π and Hind III. This is for the production of CNP (1287bp: 37-1323) and TNP (357bp: 37-393) polypeptides, the amino acid sequence of which is shown in SEQ ID NOs: 2 and 3. The plasmid into which 369bp containing TNP was inserted was named pGT-SooV2-TNP, and the plasmid into which 1299bp including CNP was inserted was named pGT-SooV2-CNP (FIG. 3).

Example 5: Cloning into Expression Vectors

PBlue-Bac-His2B (Invitrogen), an expression fusion vector containing His-Taq at the N-terminus, was treated with restriction enzymes Bgl Π and Hind III, and pGT-SooV2-TNP and pGT-SooV2-CNP PCR products were also cloned with the restriction enzymes Bgl Π and Hind III to obtain pBac-SooV2-TNP and pBac-SooV2-CNP (Fig. 4).

Example 6 Construction of Recombinant Baculovirus

pBac-SooV2-TNP and pBac-SooV2-CNP were prepared with Bac-SooV2-TNP and Bac-SooV2-CNP using Bac-N-Blue baculovirus expression vector system (FIG. 5). .

Sf9 cells ( ⁶ × ² × ¹⁰ ) in the log phase were poured into a 60 mm dish to form a monolayer. To prepare a transfection mixture, 10 ul (0.5 ug) of Bac-N-Blue ^TM DNA was added to a microcentrifuge and the following solution was added.

pBac-SooV2-TNP or pBac-SooV2-CNP (1ug / ul): 4ul

Grace's insect media (without supplements or FBS): 1 ml

Cellfectin reagent (mix well before use and always): 20 ul

After 15 minutes at room temperature, remove the medium from the dish and add 2 ml of Grace's insect media without supplements or FBS, remove the medium from the monolayer, and then prepare the transfection mixture. Put the drops in. After leaving the dish at room temperature for about 4 hours, 1 ml of TNM-FH medium was added, and the dish was sealed and incubated at 27 ° C for 72 hours. The recombinant baculovirus thus cotransfected and replicated is referred to as a virus stock.

Example 8: Recombinant Baculovirus Purification

Virus stock concentrations were measured by an end-point dilution method.

End-point dilution to determine the titer of the virus was performed by diluting the virus inoculation from 10 ^-5 to 10 ^-8 times, and dispensing at a concentration of ¹ × 10 ⁴ cells per well in 96 well plates. Each diluted solution was inoculated to the cultured cells, and the culture was continued at 27 ° C. Virus concentrations were calculated from the ratio of virus infected wells to uninfected wells to determine plaque forming units (PFUs).

For the selection of pure virus, the virus inoculum was diluted 10 ^-2 to 10 ^-8 fold, and each dilution was inoculated into cultured cells dispensed at a concentration of ¹ × 10 ⁴ cells per well in 96 well plates, and the culture was continued at 27 ° C. . Four days after the inoculation, the cells were observed under the microscope for the formation of intracellular polyhedron and virus infection, and the virus was selected in the wells of the maximum dilution factor.

Example  9: Expression and Isolation of Recombinant Proteins

Insect cells Sf9 cells were seeded about 5 X 10 ⁶ in a 75 cm 2 flask, and then inoculated with 5 MOI of recombinant virus. All cells were harvested after confirming inclusion body formation 5 days after infection. The collected cells were centrifuged at 6,000 g (7,000 rpm) to collect the salivary cells, suspended in 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl), and then subjected to ultrasonic grinding at 15 Hz at 15 Hz. Stir six times for a second. The supernatant was stored in soluble form by centrifugation at 23,400 g (14,000 rpm) for 15 minutes. Insoluble form was also suspended in 1 X binding buffer containing 6M Urea and allowed to stand at 4 ° C for 1 hour, followed by centrifugation at 23,400g (14,000rpm) for 30 minutes. lysed pellet) was collected. The harvested pellet was purified through His-bind chromatography. The soluble form, the lysed pellet, and the purified pellet were purified by SDS-PAGE to confirm the fraction of the recombinant protein in the Western blot. As a control, a soluble form of wild-type virus infected cells was used. Western blots were used as primary antibodies for patients with nephrotic bleeding (HFRS) and normal serum.

As shown in FIG. 6, it was confirmed that more protein was present in the soluble form than in the insoluble form of the TNP. In addition to the specific band of the 22 kDa TNP fraction, the bands that appear weak below them are strongly expressed by Western blot by patient serum in the purified pellet as well as the soluble form. Was estimated to be. It was also demonstrated by the absence of both bands for normal subjects. The lower band was assumed to be due to partial degradation of TNP.

7 is a Western blot of purified pellets of CNP and TNP. As shown in the results, it was confirmed that CNP responded only to patients with renal hemorrhagic fever (HFRS) and produced about 50 kDa of recombinant protein.

Example  10: His - bind  friendship Chromatography  Purification of Recombinant Proteins

After filling his column with 2 ml of His-bind resin (Novagen), the column was prepared using distilled water, 1 X charge buffer, and 1 X binding buffer. The antigenic protein was passed through a column, followed by 1 X binding buffer (5 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl) and 1 X washing buffer (40 mM imidazole, 20 mM Tris-HCl pH 7.9, 0.5 M NaCl). ). 1 ml of protein was recovered through elution buffer (300 mM imidazole, 0.5 M NaCl, 20 mM Tris-Cl pH 7.9). Protein concentration in each fraction was quantified using the Raleigh method, and the protein was confirmed by SDS-PAGE and Western blot.

Example  11: Recombinant Protein Analysis

11-1. Western - blot

Recombinant proteins purified on 10% gel of 1 mm or 1.5 mm thickness were subjected to electrophoresis at 10 ug per well. Electrophoresis gels were transferred for 40 minutes at 80 volt using a Bio-rad transport machine. The transferred membrane was blocked with 5% skim milk-TBST (Tris-buffered Saline Tween 20) for 1 hour, and the serum of the patient, the primary antibody, was diluted 1: 3000 and reacted for 1 hour. . The secondary antibody was reacted by diluting the horse-radish peroxidase conjugate anti-human IgG to 1: 10000. After the reaction was completed using the ECL kit.

As a result of Western blot, recombinant CNP and TNP responded at 50kDa for CNP and 22kDa for CNP in serum of nephrotic bleeding fever but did not react with normal serum (FIGS. 6 and 7).

11-2. Dot - blot

1 ug of recombinant protein was added dropwise to the nitrocellulose membrane and dried at 37 ° C. for 1 hour. The dried membrane was blocked for 1 hour with 5% skim milk-TBST, and the reaction was performed for 1 hour by diluting the primary antibody patient serum and normal serum 1: 3,000. The secondary antibody, Horse-radish peroxidase conjugate anti-human IgG, was diluted 1: 10,000 and reacted. After the reaction was completed using the ECL kit.

Dot-blotting was performed with serum of patients with nephrotic bleeding fever infected with Hantan virus or Seoul virus. As shown in Table 2, CNP showed a stronger positive signal than TNP, and all of them reacted with normal normal serum. Turned out not to.

Reactivities of recombinant NP antigens against HRFS patients by Dot-blots. HRFS patients
serum No. IFA value (HTN) ^a Dot-blot Value IgG TNP
(IgG) CNP
(IgG) 2001-216 1280 +++ +++ 2001-193 80 + + 2001-36 160 ++ +++ 2001-42 1280 +++ +++ 2000-449 1280 +++ +++ 99-445 640 +++ +++ 99-485 320 + ++ 99-444 640 +++ +++ 99-235 320 + ++

^a Data are presented as a strong positive (+++), middle positive (++) or scarcely positive (+) result by Dot-blot.

* Ten normal sera did not react with recombinant NP antigens

11-3. ELISA

The recombinant protein antigen was diluted in 0.05 M carbonate buffer solution (pH 9.6) to 1 ug / ml, coated on a 96 well microplate, and reacted at 4 ° C. for 18 hours. After washing twice with phosphate buffer solution (PBST) containing 0.05% Tween 20 and blocking with 3% BSA for 2 hours at 37 ℃. The primary antibody was used by diluting the serum of rats infected with Hantan, Seoul, Pummala or Prospect Hill virus, and the secondary antibody was horse-radish conjugate anti-rat. IgG (IgM) was used diluted 1: 6000. After color development with a color substrate solution, OD was measured at a wavelength of 490 nm with an ELISA reader.

As shown in Table 3, the recombinant antigen CNP responded to Hantan virus and Seoul virus, but not to Fumala virus and Prospect Hill virus.

Antigenic Characteristics of Recombinant CNP to Rat Antisera infected with Hantavirus Rat Antisera against Hantaan Seoul Pummala Prospect hill Reactivity of CNP by ELISA +++ +++ - -

As a result of testing the antigenic results of CNP and TNP, it was found that CNP is more antigenic than TNP. We have developed a diagnostic kit to check the performance of the diagnostic kit for diagnosing nephrotic bleeding hemorrhagic fever with excellent antigenic CNP.

Example 12 Lateral flow assay (Immunochromatography) (Rapid Diagnostic Kit, RDK)

12-1. Antigen Immobilization on Nitrocellulose Membranes

Immobilization of specific antigens, which serve to capture assay specific antibodies, is carried out by electrostatic interaction on a nitrocellulose (NC) membrane (5 um pore size). The lyophilized recombinant antigen was dissolved in 0.1% SDS buffer and 1 cm from the bottom of the plastic backed NC membrane strip (5 × 25 mm), and the control antibody (anti-Goat-IgG) was placed in the dispenser at 1.4 cm. (dispenser) was used to instill at a rate of 1 ul / cm. The deposited membrane was dried at 37 ° C. for 1 hour.

As a result of varying the concentration of SDS buffer and antigen of lyophilized antigen, the colored signal was maximum at the antigen concentration of 0.6 mg / ml and decreased at the concentration below or above. This is because the signal development requires a sufficient amount of antigen to bind to the analyte, but when the excess antigen is concentrated in a certain area, the spatial approach for antigen-antibody reaction is difficult, which lowers the probability of immunoadhesion.

12-2. antigen Application  buffer

① Tween 20

In the case of Tween 20, it affects the nonspecific reaction of antigenic proteins. The higher the concentration, the lower the signal strength and the addition of Tween 20 is necessary to eliminate cross-reaction. Tween 20 concentration range was tested to 0.001, 0.005, 0.01%.

② SDS

Since currently used antigen-derived antigens are expressed in the form of inclusion bodies, an amount of SDS is required to maintain the solubility of the antigenic protein. Buffer change after antigen purification has been shown to rapidly decrease solubility and thus lower antigenicity.

The surfactant contained in the antigen buffer was Tween 20, and it was found that 0.01% Tween 20 was appropriate when tested in concentration ranges of 0.001%, 0.01%, and 0.05%. In addition, an appropriate concentration of SDS is required to maintain antigen solubility. As a result of testing the SDS concentration from 0.5% to 0.01%, it was confirmed that 0.1% SDS is the proper concentration at present.

12-3. Gold-antibody polymer concentration test

The specific signal intensity generated from the assay system is increased in proportion to the amount of gold-antibody polymer used. However, when used in excess, non-specific signals are increased resulting in false positive results. In this experiment, gold conjugated goat anti-human IgG (H + L) (BBI, 40 nm) was used as a polymer, and the concentration of the gold-antibody polymer was tested between OD 2 and 6. As a result, OD 2 for IgM and OD 4 for IgG were most suitable.

12-4 strip fabrication

The system is designed with a NC backing membrane, a plastic backing reaction membrane, to immobilize the antigen in the middle, attach a glass fiber membrane (conjugate pad) where polymer is accumulated, and a cellulose membrane that absorbs excess aqueous solution. It is formed by connecting the ends (sample pad and absorbent pad) in contact with each other. The glass fiber membrane located at the bottom can absorb the sample in a short time due to its high absorption ability, and can induce an efficient reaction between the analyte and the polymer by prolonging the retention time before the dissolved polymer is transferred to the upper immune strip. On the intermediate immune strip, a test line or capture line to which an analyte measuring antigen was fixed and a control line to which a specific antibody to detect a polymer were spatially separated and immobilized. The cellulose membrane, an absorbent pad located in, allowed the excess sample to be absorbed quickly (FIG. 8).

If the patient's serum is dropped on the sample pad and observed, if it is positive (infection with pathogens), the red color on the test line can be visually observed. If negative, red color appears only at the control line.

12-5. Simultaneous Detection of IgM and IgG Rapid Diagnostic Kit Preparation

Strips for each of IgM and IgG prepared as 12-4 were cut to a width of 4 mm and a height of 60 mm, and then placed in one plastic device for simultaneous diagnosis (FIG. 9). The method of use is to dilute the patient's plasma or serum with an appropriate dilution in a small well at the bottom, drop 300ul, wait 5 minutes, and then develop red color on the reaction line (position T) and control line (position C). Observe. Both lines are positive if they develop color, negative if only the control line develops, and no color development occurs.

Example 13: Sensitivity and Specificity of Rapid Diagnostic Kit (RDK)

To examine the diagnostic sensitivity and specificity of the nephrotic bleeding fever diagnostic kit, serum of patients with nephrotic bleeding fever and serum of other acute febrile diseases (rash, leptospirosis, Tsutsugashi disease) were tested. It was.

13-1. Comparison of sensitivity and specificity of IFA and RDK

Sensitivity and specificity of the diagnostic kit (RDK) made from whole protein (CNP) for 31 samples diagnosed with hemorrhagic fever with IFA results of IgG 1:80 or higher and 10 normal patients requested by the Department of Microbiology, College of Medicine, Korea University The degree was examined.

Comparison of Hantaviral Human Antibody by IFA and RDK IFA RDK IgG (H + L) IgG IgM IgG or IgM HFRS (n = 31) 31 27 21 29 Non-HFRS (n = 10) 0 0 0 0

Serum dilution (1:80)

Sensitivity of RDK: 29/31 X 100 = 94%

As shown in Table 4, the titer criterion was 1:80 based on IFA, and the performance of RDK was examined. As a result, sensitivity of IgM and IgG was 68% and 87%, respectively. According to the test results, the sensitivity was 94% and the specificity was 100%. IFA is limited in the detection of anti-hantavirus IgM in patients with nephrotic bleeding. However, RDK suggests anti-Hantavirus IgM detection rate of 68% in serum diagnosed with HFRS patients based on IFA IgG (1:80), as shown in Table 4 and FIG. 10, for early diagnosis.

Comparison of IFA and RDK for Diagnosis of HFRS among Acute Febrile Illnesses (AFI) IFA RDK (1: 100) ^a IgG (H + L)
(1:80) ^a IgM (1:20) ^a IgG or IgM IgG IgM IgG or IgM HFRS
(n = 56) 56 21 56 48 42 55 Other AFI
(n = 150) 0 0 0 0 0 0 FUO
(n = 15) 0 0 0 One 0 One Healthy control
(n = 10) 0 0 0 0 0 0

a: dilution ratio of sera

Sensitivity: 55/56 X 100 = 98%

Specificity among other AFI & FUO (fever of unknown origin): 164/165 X 100 = 99%

As shown in Table 5, which is another experiment, the sensitivity of RDK is 98% and the specificity is 99% based on IFA. One of the fifteen patients with fever, but whose cause was unknown with IFA (FUO: fever of unknown origin), was positive in RDK. The patient may have been previously infected with nephrotic bleeding fever, or the cause of the fever may be HFRS, and RDK may not be able to diagnose it. If the patient is positive, the sensitivity of the RDK may be 100%. In addition, IgM in IFA is detected about 38%, but in the case of RDK is detected at 75%, showing a double sensitivity. This indicates that RDK has a superior early diagnosis capability compared to conventional IFA (Table 5, FIG. 11).

1 is a diagram showing the gene structure of the nucleocapsid protein, Complete Protein (CNP) and Truncated NP (TNP) in the S segment of the hearing virus.

Figure 2 is a diagram showing the DNA band (about 370bp) of the fragment protein (TNP) and the DNA band (about 1300bp) of the whole protein (CNP) after the PCR reaction.

3 is a diagram showing cloning of fragment protein (TNP) DNA and whole protein (CNP) DNA into pGem-T-Easy PCR cloning vector. pGT-SooV2-TNP refers to a plasmid in which a fragment protein (TNP) is inserted, and pGT-SooV2-CNP refers to a plasmid in which a whole protein (CNP) is inserted.

FIG. 4 is a diagram of pBac-SooV2-TNP and pBac-Soov2-CNP showing the cloning of PCR products of pGT-SooV2-TNP and pGT-SooV2-CNP in a baculovirus plasmid vector having His-Taq at the N-terminus. to be.

5 is a diagram showing the production of pBac-SooV2-TNP and pBac-SooV2-CNP with Bac-SooV2-TNP and Bac-SooV2-CNP using a baculovirus expression vector system.

Figure 6 is a diagram showing the results of Western blot using patients and normal serum for the separation detection of fragment protein (TNP). The band of the down arrow appears to be due to partial degradation of the fragment protein. The photo on the left shows the result of nephrotic hemorrhagic fever patient serum as the primary antibody and the serum on the normal subject as the primary antibody. (1 lane: solution fraction of cells infected with wild-type baculovirus, 2 lanes: lysate fraction of cells infected with recombinant baculovirus, 3 lanes: undissolved fraction of cells were treated with buffer containing urea and centrifuged). Supernatant, 4 lanes: 3)

7 is a diagram showing the results of Western blot for purified fractions of fragment protein (TNP) and whole protein (CNP). (1 lane: cell not infected with virus, 2 lane: cell infected with baculovirus, 3 lane: cell infected with recombinant baculovirus cloned with whole protein (CNP), 4 lane: fragment protein (TNP) Cells infected with the recombinant baculovirus cloned, 5 lanes: cells infected with the recombinant baculovirus cloned with whole protein (CNP), 6 lanes: infected with the recombinant baculovirus cloned with the fragment protein (TNP) cell)

8 is a diagram showing the structure of a test strip in a diagnostic kit according to the present invention prepared by introducing immunochromatography technology.

Figure 9 illustrates the structure of the final product capped with a plastic device in the diagnostic kit of the present invention.

FIG. 10 shows antibody test results by a diagnostic kit (RDK) according to the present invention in a patient (31 samples) in which IgG antibodies to nephrotic bleeding hemorrhagic fever were detected by indirect immunofluorescence antibody (IFA). In the test for IgM and IgG, the sensitivity was 68% and 87%, respectively, and the sensitivity of RDK was 94%.

11 shows the sensitivity and specificity of a diagnostic kit (RDK) according to the present invention. In addition, IgM detection rates of indirect immunofluorescent antibody (IFA) and the diagnostic kit (RDK) are compared.

<110> Immunemed Co. <120> Rapid diagnostic kit of hemorrhagic fever with renal syndrome          detecting specific IgM and IgG using nucleocapsid protein derived          Soochong virus <130> PA9608-494 / KR <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 1696 <212> DNA <213> S (small) segment of Soochong virus <400> 1 tagtagtaga ctccctaaag agctacgata caaacaatgg caactatgga ggaattgcaa 60 aaggagatca atgcccatga gggtcaactt gtgatagcaa ggcaaaaagt aagggatgca 120 gaaagccaat atgagaagga tccagatgag ttgaaccaaa gagcattaac agacagagaa 180 ggggttgcag catccattca ggcaaaaatt gatgaattaa agaggcagtt ggcagataga 240 atagcaactg gaaaaaacct gggaaaggag caagacccaa caggtgttga acccggggac 300 catttaaaag agagatcaat gctcagctat gggaatgtac tggatttgaa ccatctagac 360 attgatgagc caacaggtca gactgcagac tggctgagca ttgttgttta tcttacatcc 420 tttgtggttc cgatactcct gaaagccctt cagatgctga caacaagggg aagacagaca 480 actaaggaca ataaagggac aagaatccga ttcaaagatg acagctcttt tgaagatgtg 540 aatggaattc ggaagccgaa acatctctat gtgtctttac ctaatgcaca gtctagtatg 600 aaggcagaag agataacacc tggaaggtac aggacagcaa tctgtggatt ataccctgct 660 cagattaaag ccagacagat ggtaagccca gtcatgagtg taattggatt ccttgccttg 720 gctaaagact ggggtgatag gattgagcag tggctaagtg aaccctgtaa gctcctccca 780 gacacagcag cagtaagcct ccatggtggt cctgcaacta atagagatta tctccggcaa 840 aggcaggcag ctttaggaaa catggagact aaggagtcaa aagcaatccg ccaacatgca 900 gaagcagcag gctgtagcat gatagaagat atcgagtcac catcatccat ctgggtgttt 960 gctggggccc cagaccgatg cccaccaaca tgtttattca ttgcagggat tgcagagtta 1020 ggtgcctttt tttccatctt acaagatatg cggaatacta taaaggcatc taagacagtt 1080 ggcacatcag aggaaaagtt gagaaagaag tcatcatttt accagtctta cctcaggaga 1140 acacaatcaa tgggaataca actggaccag agaattattg ttctctttat ggttgcctgg 1200 gggaaggaag cagtggataa tttccatctt ggagatgacg tggatcctga attgaggaca 1260 ctagcacaga gtttgattga tctaaaagtg aaggaaattt caaaccaaga acctttaaaa 1320 ctataatcac tgaatgtata aatcctttta tgtgattatc atatactact gaatcattat 1380 caatcatatt tgcactatta ttatcagggg aatcagtata tcagggcatg ggaacattta 1440 tgggtgggaa tcattactca ggggtgggtc agttaatccg ttgtgggtgg gtttagctcc 1500 aggctacctt aagtagcctt tttttgtata tatggatgta gatttcattt gatccttaac 1560 taatcttgtt ttctttccct ttctttctgc tttctctgct tactaacaac aacattctac 1620 ctcaacacaa aactacctca acttaactac ctcatttgat tgctccttga ttgtcttttt 1680 agggagcata ctacta 1696 <210> 2 <211> 429 <212> PRT <213> complete nucleocapsid protein in S segment of Soochong virus <400> 2 Met Ala Thr Met Glu Glu Leu Gln Lys Glu Ile Asn Ala His Glu Gly   1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Arg Asp Ala Glu Arg Gln Tyr              20 25 30 Glu Lys Asp Pro Asp Glu Leu Asn Gln Arg Ala Leu Thr Asp Arg Glu          35 40 45 Gly Val Ala Ala Ser Ile Gln Ala Lys Ile Asp Glu Leu Lys Arg Gln      50 55 60 Leu Ala Asp Arg Ile Ala Thr Gly Lys Asn Leu Gly Lys Glu Gln Asp  65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Met Leu                  85 90 95 Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro             100 105 110 Thr Gly Gln Thr Ala Asp Trp Leu Ser Ile Val Val Tyr Leu Thr Ser         115 120 125 Phe Val Val Pro Ile Leu Leu Lys Ala Leu His Met Leu Thr Thr Arg     130 135 140 Gly Arg Gln Thr Thr Lys Asp Asn Lys Gly Thr Arg Ile Arg Phe Lys 145 150 155 160 Asp Asp Ser Ser Phe Glu Asp Val Asn Gly Ile Arg Lys Pro Lys His                 165 170 175 Leu Tyr Val Ser Leu Pro Asn Ala Gln Ser Ser Met Lys Ala Glu Glu             180 185 190 Ile Thr Pro Gly Arg Tyr Arg Thr Ala Ile Cys Gly Leu Tyr Pro Ala         195 200 205 Gln Ile Lys Ala Arg Gln Met Val Ser Pro Val Met Ser Val Ile Gly     210 215 220 Phe Leu Ala Leu Ala Lys Asp Trp Gly Asp Arg Ile Glu Gln Trp Leu 225 230 235 240 Ser Glu Pro Cys Lys Leu Leu Pro Asp Thr Ala Ala Val Ser Leu His                 245 250 255 Gly Gly Pro Ala Thr Asn Arg Asp Tyr Leu Arg Gln Arg Gln Ala Ala             260 265 270 Leu Gly Asn Met Glu Thr Lys Glu Ser Lys Ala Ile Arg Gln His Ala         275 280 285 Glu Ala Ala Gly Cys Ser Met Ile Glu Asp Ile Glu Ser Pro Ser Ser     290 295 300 Ile Trp Val Phe Ala Gly Ala Pro Asp Arg Cys Pro Pro Thr Cys Leu 305 310 315 320 Phe Ile Ala Gly Ile Ala Glu Leu Gly Ala Phe Phe Ser Ile Leu Gln                 325 330 335 Asp Met Arg Asn Thr Ile Lys Ala Ser Lys Thr Val Gly Thr Ser Glu             340 345 350 Glu Lys Leu Arg Lys Lys Ser Ser Phe Tyr Gln Ser Tyr Leu Arg Arg         355 360 365 Thr Gln Ser Met Gly Ile Gln Leu Asp Gln Arg Ile Val Leu Phe     370 375 380 Met Val Ala Trp Gly Lys Glu Ala Val Asp Asn Phe His Leu Gly Asp 385 390 395 400 Asp Val Asp Pro Glu Leu Arg Thr Leu Ala Gln Ser Leu Ile Asp Leu                 405 410 415 Lys Val Lys Glu Ile Ser Asn Gln Glu Pro Leu Lys Leu             420 425 <210> 3 <211> 119 <212> PRT <213> truncated nucleocapsid protein in S segment of Soochong virus <400> 3 Met Ala Thr Met Glu Glu Leu Gln Lys Glu Ile Asn Ala His Glu Gly   1 5 10 15 Gln Leu Val Ile Ala Arg Gln Lys Val Arg Asp Ala Glu Arg Gln Tyr              20 25 30 Glu Lys Asp Pro Asp Glu Leu Asn Gln Arg Ala Leu Thr Asp Arg Glu          35 40 45 Gly Val Ala Ala Ser Ile Gln Ala Lys Ile Asp Glu Leu Lys Arg Gln      50 55 60 Leu Ala Asp Arg Ile Ala Thr Gly Lys Asn Leu Gly Lys Glu Gln Asp  65 70 75 80 Pro Thr Gly Val Glu Pro Gly Asp His Leu Lys Glu Arg Ser Met Leu                  85 90 95 Ser Tyr Gly Asn Val Leu Asp Leu Asn His Leu Asp Ile Asp Glu Pro             100 105 110 Thr Gly Gln Thr Ala Asp Trp         115 <210> 4 <211> 28 <212> DNA <213> forward primer for complete nucleocapsid protein <400> 4 agatctaatg gcaactatgg aggaattg 28 <210> 5 <211> 30 <212> DNA <213> reverse primer for complete nucleocapsid protein <400> 5 aagcttatag ttttaaaggt tcttggtttg 30 <210> 6 <211> 28 <212> DNA <213> forward primer for truncated nucleocapsid protein <400> 6 agatctaatg gcaactatgg aggaattg 28 <210> 7 <211> 28 <212> DNA <213> reverse primer for truncated nucleocapsid protein <400> 7 aagcttacca gtctgcagtc tgacctgt 28  

Claims (4)

Renal syndrome hemorrhagic fever diagnostic composition comprising a nucleocapsid protein derived from a bovine virus having an amino acid sequence of SEQ ID NO: 2 or 3 produced from eukaryotic cells, and simultaneously detects hemorrhagic fever-specific IgM and IgG. (a) a sample pad on which a sample is absorbed; (b) gold conjugation pads that bind to human antibodies in the sample; (c) a test membrane in which a test line comprising the protein of claim 1 as an antigen and a control line comprising a control protein are treated; And (D) Nephrotic hemorrhagic fever diagnostic kit consisting of a test strip comprising an absorption pad (absorption pad) to absorb the remaining amount of the sample, and simultaneously detects nephrotic hemorrhagic fever-specific IgM and IgG. delete A method for detecting an antibody against a hantan virus of nephrotic bleeding fever using the kit of claim 2.

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